Lactase, also known as β-galactosidase (EC 3.2.1.23), the main function is to hydrolyze lactose to glucose and galactose. Lactase is a non-toxic biological enzyme preparation that has been identified as a safe substance by the FDA and other regulatory authorities, allowing the use in the food industrial production.
Sources
Native lactases are mainly found in yeast, bacteria, plants, and the gut of young animals. Many microorganisms can produce lactases, including bacteria (Lactobacillus, Bacillus coli, Lactobacillus, and amyloliquefaciens), molds (Aspergillus oryzae, Aspergillus niger, and Aspergillus globosum), yeast (Kluyveromyces and Kluyveromyces lactis), Actinomyces (azure Streptococcus) and so on.
Properties
Lactase is a white powder with odorless. After dissolving, lactase can become a light brown liquid. Lactose has low solubility that can be easily precipitated in cold animal products, making the products with a granular structure. Optimum pH of lactase range is 7.0 to 7.5 from E. coli, 6.0 to 7.0 from yeast, about 5.0 from mold. The optimum temperature range is 37 to 50°C. In normal use concentrations, lactase can hydrolyze 74% of lactose in 72 hours.
Catalytic Mechanism
In metabolism, the β-glycosidic bond of D-lactose is hydrolyzed by lactase to form D-galactose and D-glucose, which can be absorbed through the intestinal walls and into the bloodstream. The catalytic mechanism of D-lactose hydrolysis retains the substrate anomeric configuration in the products. Although the details of the mechanism are uncertain, the stereochemical retention is achieved by a double displacement reaction. Studies of lactase have proposed that hydrolysis is initiated when a glutamate nucleophile on the enzyme attacks from the axial side of the galactosyl carbon in the β-glycosidic bond. Mg-dependent acid catalysis can facilitate the removal of the D-glucose leaving group. The enzyme is liberated from the α-galactosyl moiety upon equatorial nucleophilic attack by water, which produces D-galactose.
Substrate modification studies have demonstrated that the 3’-OH and 2’-OH moieties on the galactopyranose ring are essential for enzymatic recognition and hydrolysis. The 3’-hydroxy group is involved in initial binding to the substrate while the 2’- group is not necessary for recognition but needed in subsequent steps. This is demonstrated by the fact that a 2-deoxy analog is an effective competitive inhibitor. Elimination of specific hydroxyl groups on the glucopyranose moiety does not completely eliminate catalysis.
Biological Functions
Lactase can break down the excessive lactose into glucose and galactose in humans. Glucose is the energy source of metabolism. Galactose is an essential structural saccharide for the metabolism of human brain and mucosal tissue. In addition, lactase can also produce oligosaccharides in the body by transglycosylation. Oligosaccharide is a dietary fiber with low molecular weight, non-stick and water-soluble characteristics, which is only used by Bifidobacteria as a growth factor in the gut while not used by spoiled bacteria. It can greatly reduce the production of harmful toxins in the intestine to prevent constipation and diarrhea.
Production
For lactase production, genetic engineering can transduce highly active lactase gene into microorganisms that are easily to culture with rapid growth and reproduction to greatly reduce costs. In order to improve the quantity and quality of the enzyme production, site-directed mutagenesis, protoplast fusion and DNA recombination technology are often used. For instance, mutagenesis of Bifidobacterium, Lactobacillus, and Streptococcus thermophilus using chemical mutagens to screen high-yielding strains. Compared with their wild-type strains, the amount of lactase produced by the mutagenized strain increased significantly. In order to further obtain high-yield strains, new genetic engineering bacteria continue to emerge. Main genetic engineering microorganisms include Escherichia coli, yeast, Streptococcus thermophiles and Lactobacillus.
Immobilized enzymes are also commonly used for lactase production. Compared with free enzymes, immobilized enzymes have many advantages. For example, immobilized enzyme is very easy to separate from substrates and products; it can be used to catalyze reactions repeatedly in a long time; it has a certain of the mechanical strength that can be used continuously in the column reactor; it is suitable for large-scale industrial production; it not only reduce the catalytic costs, but also can greatly reduce the biocatalyst manufacturing waste emissions and operational pollution. Enzyme immobilization methods include embedding method, adsorption method, covalent binding method, etc.
Applications
Lactase is mainly used in the dairy industry to convert light sweetness and low-solubility lactose to sweeter and more soluble monosaccharides (glucose and galactose). Lactase can also reduce the possibility of crystallization of lactose in ice cream, concentrated milk, and light condensed milk, while increasing sweetness. In the fermentation and bakery industry, lactase can be used by yeast to hydrolyze lactose to glucose. There are a number of babies due to the lack of normal lactase in the intestine resulting diarrhea after feeding milk, so many countries in Europe often add lactase and lysozyme to milk for infants.
